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Antimicrobial resistance (AMR) is rapidly becoming one of the great healthcare challenges. A common mechanism employed by pathogenic bacteria to avoid the action of certain antibiotics is to overexpress efflux pumps that can extrude these drugs from the cell rendering them ineffective. Small molecule inhibitors that target bacterial efflux pumps provide a route toward reversing AMR. Here, we describe the application of surface plasmon resonance (SPR) technology to characterize protein:small molecule interactions between the inner membrane protein AcrB subunit of the Escherichia coli AcrA-AcrB-TolC efflux pump and its substrates and novel inhibitors. The SPR assay provides quantitative data about the kinetics of binding that can help guide the development of new chemotherapies to combat AMR. Refereed/Peer-reviewed

usc Drug efflux pumps confer multidrug resistance to dangerous bacterial pathogens which makes these proteins promising drug targets. Herein, we present initial chemical optimization and structure-activity relationship (SAR) data around a previously described efflux pump inhibitor, nordihydroguaretic acid (NDGA). Four series of novel NDGA analogues that target Escherichia coli AcrB were designed, synthesized and evaluated for their ability to potentiate the activity of antibiotics, to inhibit AcrB-mediated substrate efflux and reduce off-target activity. Nine novel structures were identified that increased the efficacy of a panel of antibiotics, inhibited drug efflux and reduced permeabilization of the bacterial outer and inner membranes. Among them, WA7, WB11 and WD6 possessing broad-spectrum antimicrobial sensitization activity were identified as NDGA analogues with favorable properties as potential AcrB inhibitors,demonstrating moderate improvement in potency as compared to NDGA. In particular, WD6 was the most broadly active analogue improving the activity of all four classes of antibacterials tested. Refereed/Peer-reviewed

A series of novel 5-methoxy-2,3-naphthalimide derivatives were designed, synthesized and evaluated for their biological activities. In particular, the ability of the compounds to synergize with antimicrobials, to inhibit Nile Red efflux, and to target AcrB was assayed. The results showed that the most of the tested compounds more sensitized the Escherichia coli BW25113 to the antibiotics than the parent compounds 7c and 15, which were able to inhibit Nile Red efflux. Significantly, compound A5 possessed the most potent antibacterial synergizing activity in combination with levofloxacin by 4 times and 16 times at the concentration of 8 and 16 µg/mL, respectively, whilst A5 could effectively abolish Nile Red efflux at 100 μM. Additionally, target effect of A5 was confirmed in the outer- or inner membrane permeabilization assays. Therefore, A5 is an excellent lead compound for further structural optimization. Refereed/Peer-reviewed

Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl β-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain. Refereed/Peer-reviewed

A novel series of 4-substituted 2-naphthamide derivatives were designed, synthesized and evaluated for their biological activity. In particular, the ability of the compounds to potentiate the action of antibiotics, to inhibit Nile Red efflux and to target AcrB specifically was investigated. The results indicated that most of the 4-substituted 2-naphthamide derivatives were able to synergize with the antibiotics tested, and inhibit Nile Red efflux by AcrB in the resistant phenotype. Subsequent exclusion of compounds with off target effects such as outer- or inner membrane permeabilization identified compounds 7c, 7g, 12c, 12i and 13g as efflux pump inhibitors (EPIs). Particularly, compounds 7c, 7g and 12i were found to be the most potent EPIs, which synergized with the two substrates tested at lower concentrations than that of parent A3, demonstrating an improvement in potency as compared to A3. Additionally, when the outer membrane of E. coli was permeabilized, compound 12c displayed a huge increase in efficacy and was able to synergize with erythromycin at a concentration that was 16 times lower than that of the parent A3. Hence we were able to design and synthesize compounds that displayed significant increase in efficacy as EPIs against AcrB. Refereed/Peer-reviewed

Drug efflux pumps confer multidrug resistance to dangerous pathogens which makes these pumps important drug targets. We have synthesised a novel series of compounds based on a 2-naphthamide pharmacore aimed at inhibiting the efflux pumps from Gram-negative bacteria. The archeatypical transporter AcrB from Escherichia coli was used as model efflux pump as AcrB is widely conserved throughout Gram-negative organisms. The compounds were tested for their antibacterial action, ability to potentiate the action of antibiotics and for their ability to inhibit Nile Red efflux by AcrB. None of the compounds were antimicrobial against E. coli wild type cells. Most of the compounds were able to inhibit Nile Red efflux indicating that they are substrates of the AcrB efflux pump. Three compounds were able to synergise with antibiotics and reverse resistance in the resistant phenotype. Compound A3, 4-(isopentyloxy)-2-naphthamide, reduced the MICs of erythromycin and chloramphenicol to the MIC levels of the drug sensitive strain that lacks an efflux pump. A3 had no effect on the MIC of the non-substrate rifampicin indicating that this compound acts specifically through the AcrB efflux pump. A3 also does not act through non-specific mechanisms such as outer membrane or inner membrane permeabilisation and is not cytotoxic against mammalian cell lines. Therefore, we have designed and synthesised a novel chemical compound with great potential to further optimisation as inhibitor of drug efflux pumps. Refereed/Peer-reviewed

Drug efflux protein complexes confer multidrug resistance on bacteria by transporting a wide spectrum of structurally diverse antibiotics. Moreover, organisms can only acquire resistance in the presence of an active efflux pump. The substrate range of drug efflux pumps is not limited to antibiotics, but it also includes toxins, dyes, detergents, lipids and molecules involved in quorum sensing; hence efflux pumps are also associated with virulence and biofilm formation. Inhibitors of efflux pumps are therefore attractive compounds to reverse multidrug resistance and to prevent the development of resistance in clinically relevant bacterial pathogens. Recent successes on the structure determination and functional analysis of the AcrB and MexB components of the AcrAB-TolC and MexAB-OprM drug efflux systems as well as the structure of the fully assembled, functional triparted AcrAB-TolC complex significantly contributed to our understanding of the mechanism of substrate transport and the options for inhibition of efflux. These data, combined with the well-developed methodologies for measuring efflux pump inhibition, could allow the rational design and subsequent experimental verification of potential efflux pump inhibitors. In this review we will explore how the available biochemical and structural information can be translated into the discovery and development of new compounds that could reverse drug resistance in Gram-negative pathogens. The current literature on efflux pump inhibitors will also be analysed and the reasons why no compounds have yet progressed into clinical use will be explored.